The present invention relates to novel compounds that are useful for inhibition and prevention of cell adhesion and cell adhesion-mediated pathologies. This invention also relates to pharmaceutical formulations comprising these compounds and methods of using them for inhibition and prevention of cell adhesion and cell adhesion-mediated pathologies. The compounds and pharmaceutical compositions of this invention can be used as therapeutic or prophylactic agents. They are particularly well-suited for treatment of many inflammatory and autoimmune diseases.
Cell adhesion is a process by which cells associate with each other, migrate towards a specific target or localize within the extra-cellular matrix. As such, cell adhesion constitutes one of the fundamental mechanisms underlying numerous biological phenomena. For example, cell adhesion is responsible for the adhesion of hematopoietic cells to endothelial cells and the subsequent migration of those hemopoietic cells out of blood vessels and to the site of injury. As such, cell adhesion plays a role in pathologies such as inflammation and immune reactions in mammals.
Investigations into the molecular basis for cell adhesion have revealed that various cell-surface macromoleculesxe2x80x94collectively known as cell adhesion molecules or receptorsxe2x80x94mediate cell-cell and cell-matrix interactions. For example, proteins of the superfamily called xe2x80x9cintegrinsxe2x80x9d are key mediators in adhesive interactions between hematopoietic cells and their microenvironment (M. E. Hemler, xe2x80x9cVLA Proteins in the Integrin Family: Structures, Functions, and Their Role on Leukocytes.xe2x80x9d, Ann. Rev. Immunol., 8, p. 365 (1990)). Integrins are non-covalent heterodimeric complexes consisting of two subunits called xcex1 and xcex2. There are at least 12 different xcex1 subunits (xcex11-xcex16, xcex1-L, xcex1-M, xcex1-X, xcex1-IIB, xcex1-V and xcex1-E) and at least 9 different xcex2 (xcex21-xcex29) subunits. Based on the type of its xcex1 and xcex2 subunit components, each integrin molecule is categorized into a subfamily.
xcex14xcex21 integrin, also known as very late antigen-4 (xe2x80x9cVLA-4xe2x80x9d), CD49d/CD29, is a leukocyte cell surface receptor that participates in a wide variety of both cell-cell and cell-matrix adhesive interactions (M. E. Hemler, Ann. Rev. Immunol., 8, p. 365 (1990)). It serves as a receptor for the cytokine-inducible endothelial cell surface protein, vascular cell adhesion molecule-1 (xe2x80x9cVCAM-1xe2x80x9d), as well as to the extracellular matrix protein fibronectin (xe2x80x9cFNxe2x80x9d) (Ruegg et al., J. Cell Biol., 177, p. 179 (1991); Wayner et al., J. Cell Biol., 105, p. 1873 (1987); Kramer et al., J. Biol. Chem., 264, p. 4684 (1989); Gehlsen et al. Science, 24, p. 1228 (1988)). Anti-VLA4 monoclonal antibodies (xe2x80x9cmAb""sxe2x80x9d) have been shown to inhibit VLA4-dependent adhesive interactions both in vitro and in vivo (Ferguson et al. Proc. Natl. Acad. Sci., 88, p. 8072 (1991); Ferguson et al., J. Immunol., 150, p. 1172 (1993)). Results of in vivo experiments suggest that this inhibition of VLA-4-dependent cell adhesion may prevent or inhibit several inflammatory and autoimmune pathologies (R. L. Lobb et al., xe2x80x9cThe Pathophysiologic Role of xcex14 Integrins In Vivoxe2x80x9d, J. Clin. Invest., 94, pp. 1722-28 (1994)).
In order to identify the minimum active amino acid sequence necessary to bind VLA-4, Komoriya et al. (xe2x80x9cThe Minimal Essential Sequence for a Major Cell Type-Specific Adhesion Site (CS1) Within the Alternatively Spliced Type III Connecting Segment Domain of Fibronectin Is Leucine-Aspartic Acid-Valinexe2x80x9d, J. Biol. Chem., 266 (23), pp. 15075-79 (1991)) synthesized a variety of overlapping peptides based on the amino acid sequence of the CS-1 region (the VLA-4 binding domain) of a particular species of fibronectin. They identified an 8-amino acid peptide, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr [SEQ ID NO: 1], as well as two smaller overlapping pentapeptides, Glu-Ile-Leu-Asp-Val [SEQ ID NO: 2] and Leu-Asp-Val-Pro-Ser [SEQ ID NO: 3], that possessed inhibitory activity against FN-dependent cell adhesion. These results suggested the tripeptide Leu-Asp-Val as a minimum sequence for cell-adhesion activity. It was later shown that Leu-Asp-Val binds only to lymphocytes that express an activated form of VLA-4, thus bringing into question the utility of such a peptide in vivo (E. A. Wayner et al., xe2x80x9cActivation-Dependent Recognition by Hematopoietic Cells of the LDV Sequence in the V Region of Fibronectinxe2x80x9d, J. Cell. Biol., 116(2), pp. 489-497 (1992)). However, certain larger peptides containing the LDV sequence were subsequently shown to be active in vivo [T. A. Ferguson et al., xe2x80x9cTwo Integrin Binding Peptides Abrogate T-cell-Mediated Immune Responses In Vivoxe2x80x9d, Proc. Natl. Acad. Sci. USA, 88, pp. 8072-76 (1991); and S. M. Wahl et al., xe2x80x9cSynthetic Fibronectin Peptides Suppress Arthritis in Rats by Interrupting Leukocyte Adhesion and Recruitmentxe2x80x9d, J. Clin. Invest., 94, pp. 655-62 (1994)].
A cyclic pentapeptide, Arg-Cys-Asp-TPro-Cys. (wherein TPro denotes 4-thioproline), which can inhibit both VLA-4 and VLA-5 adhesion to FN has also been described (D. M. Nowlin et al. xe2x80x9cA Novel Cyclic Pentapeptide Inhibits xcex14xcex21 and xcex15xcex21 Integrin-mediated Cell Adhesionxe2x80x9d, J. Biol. Chem., 268(27), pp. 20352-59 (1993); and PCT publication PCT/US91/04862). This peptide was based on the tripeptide sequence Arg-Gly-Asp from FN which had been known as a common motif in the recognition site for several extracellular-matrix proteins.
Despite these advances, there remains a need for small, specific inhibitors of VLA-4-dependent cell adhesion. Ideally, such inhibitors would be semi-peptidic or non-peptidic so that they may be orally administered. Such compounds would provide useful agents for treatment, prevention or suppression of various pathologies mediated by cell adhesion and VLA-4 binding.
The present invention solves this problem by providing novel non-peptidic compounds that specifically inhibit the binding of ligands to VLA-4. These compounds are useful for inhibition, prevention and suppression of VLA-4-mediated cell adhesion and pathologies associated with that adhesion, such as inflammation and immune reactions. The compounds of this invention may be used alone or in combination with other therapeutic or prophylactic agents to inhibit, prevent or suppress cell adhesion. This invention also provides pharmaceutical formulations containing these VLA-4-mediated cell adhesion inhibitors and methods of using the compounds and compositions of the invention for inhibition of cell adhesion.
According to one embodiment of this invention, these novel compounds, compositions and methods are advantageously used to treat inflammatory and immune diseases. The present invention also provides methods for preparing the compounds of this invention and intermediates useful in those methods.
Definitions
As used herein, the term xe2x80x9calkylxe2x80x9d, alone or in combination, refers to a straight-chain or branched-chain alkyl radical containing from 1 to 10, preferably from 1 to 6 and more preferably from 1 to 4, carbon atoms. Examples of such radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, decyl and the like.
The term xe2x80x9calkenylxe2x80x9d, alone or in combination, refers to a straight-chain or branched-chain alkenyl radical containing from 2 to 10, preferably from 2 to 6 and more preferably from 2 to 4, carbon atoms. Examples of such radicals include, but are not limited to, ethenyl, E- and Z-propenyl, isopropenyl, E- and Z-butenyl, E- and Z-isobutenyl, E- and Z-pentenyl, decenyl and the like.
The term xe2x80x9calkynylxe2x80x9d, alone or in combination, refers to a straight-chain or branched-chain alkynyl radical containing from 2 to 10, preferably from 2 to 6 and more preferably from 2 to 4, carbon atoms. Examples of such radicals include, but are not limited to, ethynyl (acetylenyl), propynyl, propargyl, butynyl, hexynyl, decynyl and the like.
The term xe2x80x9ccycloalkylxe2x80x9d, alone or in combination, refers to a cyclic alkyl radical containing from 3 to 8, preferably from 3 to 6, carbon atoms. Examples of such cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.
The term xe2x80x9ccycloalkenylxe2x80x9d, alone or in combination, refers to a cyclic carbocycle containing from 4 to 8, preferably 5 or 6, carbon atoms and one or more double bonds. Examples of such cycloalkenyl radicals include, but are not limited to, cyclopentenyl, cyclohexenyl, cyclopentadienyl and the like.
The term xe2x80x9carylxe2x80x9d refers to a carbocyclic aromatic group selected from the group consisting of phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl, and anthracenyl; or a heterocyclic aromatic group selected from the group consisting of furyl, thienyl, pyridyl, pyrrolyl, oxazolyly, thiazolyl, imidazolyl, pyrazolyl, 2-pyrazolinyl, pyrazolidinyl, isoxazolyl, isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl, 1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, 1,3,5-trithianyl, indolizinyl, indolyl, isoindolyl, 3H-indolyl, indolinyl, benzo[b]furanyl, 2,3-dihydrobenzofuranyl, benzo[b]thiophenyl, 1H-indazolyl, benzimidazolyl, benzthiazolyl, purinyl, 4H-quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl.
xe2x80x9cArylxe2x80x9d groups, as defined in this application may independently contain one to three substituents which are independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, cyano, carboxy, carboalkoxy, Arxe2x80x2-substituted alkyl, Arxe2x80x2-substituted alkenyl or alkynyl, 1,2-dioxymethylene, 1,2-dioxyethylene, alkoxy, alkenoxy or alkynoxy, Arxe2x80x2-substituted alkoxy, Arxe2x80x2-substituted alkenoxy or alkynoxy, alkylamino, alkenylamino or alkynylamino, Arxe2x80x2-substituted alkylamino, Arxe2x80x2-substituted alkenylamino or alkynylamino, Arxe2x80x2-substituted carbonyloxy, alkylcarbonyloxy, aliphatic or aromatic acyl, Arxe2x80x2-substituted acyl, Arxe2x80x2-substituted alkylcarbonyloxy, Arxe2x80x2-substituted carbonylamino, Arxe2x80x2-substituted amino, Arxe2x80x2-substituted oxy, Arxe2x80x2-substituted carbonyl, alkylcarbonylamino, Arxe2x80x2-substituted alkylcarbonylamino, alkoxy-carbonylamino, Arxe2x80x2-substituted alkoxycarbonyl-amino, Arxe2x80x2-oxycarbonylamino, alkylsulfonylamino, mono- or bis-(Arxe2x80x2-sulfonyl)amino, Arxe2x80x2-substituted alkyl-sulfonylamino, morpholinocarbonylamino, thiomorpholinocarbonylamino, N-alkyl guanidino, Nxe2x80x94Arxe2x80x2 guanidino, Nxe2x80x94Nxe2x80x94(Arxe2x80x2,alkyl) guanidino, N,Nxe2x80x94(Arxe2x80x2,Arxe2x80x2)guanidino, N,N-dialkyl guanidino, N,N,N-trialkyl guanidino, N-alkyl urea, N,N-dialkyl urea, Nxe2x80x94Arxe2x80x2 urea, N,Nxe2x80x94(Arxe2x80x2,alkyl) urea and N,Nxe2x80x94(Arxe2x80x2)2 urea; wherein xe2x80x9cArxe2x80x2xe2x80x9d is a carbocyclic or heterocyclic aryl group as defined above having one to three substituents selected from the group consisting of hydrogen, halogen, hydroxyl, amino, nitro, trifluoromethyl, trifluoromethoxy, alkyl, alkenyl, alkynyl, 1,2-dioxymethylene, 1,2-dioxyethylene, alkoxy, alkenoxy, alkynoxy, alkylamino, alkenylamino or alkynylamino, alkylcarbonyloxy, aliphatic or aromatic acyl, alkylcarbonylamino, alkoxycarbonylamino, alkylsulfonylamino, N-alkyl or N,N-dialkyl urea.
The term xe2x80x9calkoxyxe2x80x9d, alone or in combination, refers to an alkyl ether radical, wherein the term xe2x80x9calkylxe2x80x9d is as defined above. Examples of suitable alkyl ether radicals include, but are not limited to, methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy and the like.
The term xe2x80x9calkenoxyxe2x80x9d, alone or in combination, refers to a radical of formula alkenyl-Oxe2x80x94, wherein the term xe2x80x9calkenylxe2x80x9d is as defined above provided that the radical is not an enol ether. Examples of suitable alkenoxy radicals include, but are not limited to, allyloxy, E- and Z-3-methyl-2-propenoxy and the like.
The term xe2x80x9calkynyloxyxe2x80x9d, alone or in combination, refers to a radical of formula alkynyl-Oxe2x80x94, wherein the term xe2x80x9calkynylxe2x80x9d is as defined above provided that the radical is not an ynol ether. Examples of suitable alkynoxy radicals include, but are not limited to, propargyloxy, 2-butynyloxy and the like.
The term xe2x80x9cthioalkoxyxe2x80x9d refers to a thioether radical of formula alkyl-Sxe2x80x94, wherein alkyl is as defined above.
The term xe2x80x9calkylaminoxe2x80x9d, alone or in combination, refers to a mono- or di-alkyl-substituted amino radical (i.e., a radical of formula alkyl-NHxe2x80x94 or (alkyl)2xe2x80x94Nxe2x80x94), wherein the term xe2x80x9calkylxe2x80x9d is as defined above. Examples of suitable alkylamino radicals include, but are not limited to, methylamino, ethylamino, propylamino, isopropylamino, t-butylamino, N,N-diethylamino and the like.
The term xe2x80x9calkenylaminoxe2x80x9d, alone or in combination, refers to a radical of formula alkenyl-NHxe2x80x94 or (alkenyl)2Nxe2x80x94, wherein the term xe2x80x9calkenylxe2x80x9d is as defined above, provided that the radical is not an enamine. An example of such alkenylamino radicals is the allylamino radical.
The term xe2x80x9calkynylaminoxe2x80x9d, alone or in combination, refers to a radical of formula alkynyl-NHxe2x80x94 or (alkynyl)2Nxe2x80x94, wherein the term xe2x80x9calkynylxe2x80x9d is as defined above, provided that the radical is not an ynamine. An example of such alkynylamino radicals is the propargyl amino radical.
The term xe2x80x9caryloxyxe2x80x9d, alone or in combination, refers to a radical of formula aryl-Oxe2x80x94, wherein aryl is as defined above. Examples of aryloxy radicals include, but are not limited to, phenoxy, naphthoxy, pyridyloxy and the like.
The term xe2x80x9carylaminoxe2x80x9d, alone or in combination, refers to a radical of formula aryl-NHxe2x80x94, wherein aryl is as defined above. Examples of arylamino radicals include, but are not limited to, phenylamino (anilido), naphthylamino, 2-, 3- and 4-pyridylamino and the like.
The term xe2x80x9cbiarylxe2x80x9d, alone or in combination, refers to a radical of formula aryl-aryl-, wherein the term xe2x80x9carylxe2x80x9d is as defined above.
The term xe2x80x9cthioarylxe2x80x9d, alone or in combination, refers to a radical of formula aryl-Sxe2x80x94, wherein the term xe2x80x9carylxe2x80x9d is as defined above. An example of a thioaryl radical is the thiophenyl radical.
The term xe2x80x9caryl-fused cycloalkylxe2x80x9d, alone or in combination, refers to a cycloalkyl radical which shares two adjacent atoms with an aryl radical, wherein the terms xe2x80x9ccycloalkylxe2x80x9d and xe2x80x9carylxe2x80x9d are as defined above. An example of an aryl-fused cycloalkyl radical is the benzo-fused cyclobutyl radical.
The term xe2x80x9caliphatic acylxe2x80x9d, alone or in combination, refers to radicals of formula alkyl-COxe2x80x94, alkenyl-COxe2x80x94 and alkynyl-CO-derived from an alkane-, alkene- or alkyncarboxylic acid, wherein the terms xe2x80x9calkylxe2x80x9d, xe2x80x9calkenylxe2x80x9d and xe2x80x9calkynylxe2x80x9d are as defined above. Examples of such aliphatic acyl radicals include, but are not limited to, acetyl, propionyl, butyryl, valeryl, 4-methylvaleryl, acryloyl, crotyl, propiolyl, methylpropiolyl and the like.
The term xe2x80x9caromatic acylxe2x80x9d, alone or in combination, refers to a radical of formula aryl-COxe2x80x94, wherein the term xe2x80x9carylxe2x80x9d is as defined above. Examples of suitable aromatic acyl radicals include, but are not limited to, benzoyl, 4-halobenzoyl, 4-carboxybenzoyl, naphthoyl, pyridylcarbonyl and the like.
The terms xe2x80x9cmorpholinocarbonylxe2x80x9d and xe2x80x9cthiomorpholinocarbonylxe2x80x9d, alone or in combination with other terms, refer to an N-carbonylated morpholino and an N-carbonylated thiomorpholino radical, respectively.
The term xe2x80x9calkylcarbonylaminoxe2x80x9d, alone or in combination, refers to a radical of formula alkyl-CONH, wherein the term xe2x80x9calkylxe2x80x9d is as defined above.
The term xe2x80x9calkoxycarbonylaminoxe2x80x9d, alone or in combination, refers to a radical of formula alkyl-OCONHxe2x80x94, wherein the term xe2x80x9calkylxe2x80x9d is as defined above.
The term xe2x80x9calkylsulfonylaminoxe2x80x9d, alone or in combination, refers to a radical of formula alkyl-SO2NHxe2x80x94, wherein the term xe2x80x9calkylxe2x80x9d is as defined above.
The term xe2x80x9carylsulfonylaminoxe2x80x9d, alone or in combination, refers to a radical of formula aryl-SO2NHxe2x80x94, wherein the term xe2x80x9carylxe2x80x9d is as defined above.
The term xe2x80x9cN-alkylureaxe2x80x9d, alone or in combination, refers to a radical of formula alkyl-NHxe2x80x94COxe2x80x94NHxe2x80x94, wherein the term xe2x80x9calkylxe2x80x9d is as defined above.
The term xe2x80x9cN-arylureaxe2x80x9d, alone or in combination, refers to a radical of formula aryl-NHxe2x80x94COxe2x80x94NHxe2x80x94, wherein the term xe2x80x9carylxe2x80x9d is as defined above.
The term xe2x80x9chalogenxe2x80x9d means fluorine, chlorine, bromine and iodine.
The term xe2x80x9cleaving groupxe2x80x9d generally refers to groups readily displaceable by a nucleophile, such as an amine, and alcohol or a thiol nucleophile. Such leaving groups are well known and include carboxylates, N-hydroxysuccinimide, N-hydroxybenzotriazole, halogen (halides), triflates, tosylates, mesylates, alkoxy, thioalkoxy and the like.
The terms xe2x80x9cactivated derivative of a suitably protected xcex1-amino acidxe2x80x9d and xe2x80x9cactivated substituted-phenylacetic acid derivativexe2x80x9d refer to the corresponding acyl halides (e.g. acid fluoride, acid chloride and acid bromide), corresponding activated esters (e.g. nitrophenyl ester, the ester of 1-hydroxybenzotriazole, HOBT, or the ester of hydroxysuccinimide, HOSu), and other conventional derivatives within the skill of the art.
In view of the above definitions, other chemical terms used throughout this application can be easily understood by those of skill in the art. Terms may be used alone or in any combination thereof. The preferred and more preferred chain lengths of the radicals apply to all such combinations.
This invention provides compounds which are capable of inhibiting VLA-4-mediated cell adhesion by inhibiting the binding of ligands to that receptor. These compounds are represented by formula (I): 
and pharmaceutically acceptable derivatives thereof;
wherein:
X is selected from the group consisting of xe2x80x94CO2H, xe2x80x94PO3xe2x88x92H, xe2x80x94SO2R5, xe2x80x94SO3H and xe2x80x94OPO3xe2x88x92H;
wherein R5 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, aryl-substituted alkyl, and aryl-substituted alkenyl or alkynyl;
Y is selected from the group consisting of xe2x80x94COxe2x80x94, xe2x80x94SO2xe2x80x94 and xe2x80x94PO2xe2x80x94;
R1 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, aryl-fused cycloalkyl, cycloalkenyl, aryl, aryl-substituted alkyl (xe2x80x9caralkylxe2x80x9d), aryl-substituted alkenyl or alkynyl, cycloalkyl-substituted alkyl, cycloalkenyl-substituted cycloalkyl, biaryl, alkoxy, alkenoxy, alkynoxy, aryl-substituted alkoxy (xe2x80x9caralkoxyxe2x80x9d), aryl-substituted alkenoxy or alkynoxy, alkylamino, alkenylamino or alkynylamino, aryl-substituted alkylamino, aryl-substituted alkenylamino or alkynylamino, aryloxy, arylamino, N-alkylurea-substituted alkyl, N-arylurea-substituted alkyl, alkylcarbonylamino-substituted alkyl, aminocarbonyl-substituted alkyl;
R2 is selected from the group consisting of hydrogen, aryl, alkyl, alkenyl or alkynyl, cycloalkyl, cycloalkenyl, and aryl-substituted alkyl;
R3 is selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aralkyl, aryl-substituted alkenyl or alkynyl, hydroxy-substituted alkyl, alkoxy-substituted alkyl, aralkoxy-substituted alkyl, amino-substituted alkyl, (aryl-substituted alkyloxycarbonylamino)-substituted alkyl, thiol-substituted alkyl, alkylsulfonyl-substituted alkyl, (hydroxy-substituted alkylthio)-substituted alkyl, thioalkoxy-substituted alkyl, acylamino-substituted alkyl, alkylsulfonylamino-substituted alkyl, arylsulfonylamino-substituted alkyl, morpholino-alkyl, thiomorpholino-alkyl, morpholino carbonyl-substituted alkyl, thiomorpholinocarbonyl-substituted alkyl, [N-(alkyl, alkenyl or alkynyl)- or N,N-[dialkyl, dialkenyl, dialkynyl or (alkyl, alkenyl)-amino]carbonyl-substituted alkyl, carboxyl-substituted alkyl, and amino acid side chains selected from arginine, asparagine, glutamine, S-methyl cysteine, methionine and corresponding sulfoxide and sulfone derivatives thereof, glycine, leucine, isoleucine, allo-isoleucine, tert-leucine, norleucine, phenylalanine, tyrosine, tryptophan, proline, alanine, ornithine, histidine, glutamine, valine, threonine, serine, aspartic acid, beta-cyanoalanine, and allothreonine;
R4 is selected from the group consisting of aryl, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl and aryl-substituted alkyl; and
n is 0, 1 or 2.
A xe2x80x9cpharmaceutically acceptable derivativexe2x80x9d denotes any pharmaceutically acceptable salt, ester, salt of such ester, amide or salt of such amide of a compound of this invention. The invention also includes any other compound which, upon administration to a patient, is capable of providing (directly or indirectly) a compound of this invention (e.g. a prodrug). The invention also includes metabolites or residues of a compound of this invention characterized by the ability to inhibit, prevent or suppress cell adhesion and cell adhesion-mediated pathologies.
In another preferred embodiment of this invention, R1 is selected from the group consisting of benzyloxy, cyanomethyl, cyclohexylmethyl, methyl, n-hexyl, N-phenylamino, phenyl, phenylcarbonyl, phenylmethyl, t-butoxy, t-butylamino, 1-indanyl, 1-naphthylmethyl, 1-phenylcyclopropyl, 2-(4-hydroxy-phenyl)ethyl, 2-(benzyloxycarbonylamino)-phenylmethyl, 2-(bis(phenylsulfonyl)amino)-phenylmethyl, 2-(Nxe2x80x2-phenylurea)phenylmethyl, 2-aminophenylmethyl, 2-benzamidophenylmethyl, 2-bromo-4-hydroxy-5-methoxyphenylmethyl, 2-hydroxyphenylmethyl, 2-naphthylmethyl, 2-phenylethyl, 2-pyridylmethyl, 2-quinolinyl, 2-[4-(Nxe2x80x2-phenylurea)phenyl]-ethyl, 3-(benzyloxycarbonylamino)-phenylmethyl, 3-(Nxe2x80x2-phenylurea)-phenylmethyl, 3-(Nxe2x80x2-phenylurea)propyl, 3-(phenylsulfonamido)-phenylmethyl, 3-acetamidophenyl-methyl, 3-aminophenylmethyl, 3-benzamidophenylmethyl, 3-hydroxy-4-(Nxe2x80x2-phenylurea)-phenylmethyl, 3-hydroxyphenylmethyl, 3indolyl, 3-methoxy-4-(Nxe2x80x2-phenylurea)-phenylmethyl, 3-methoxy-4-(Nxe2x80x2-(2-methylphenyl)urea)-phenylmethyl, 3-methyl-4-(Nxe2x80x2-phenylurea)-phenylmethyl, 3-nitrophenylmethyl, 3-phenylpropyl, 3-pyridylmethyl, 4-(2-aminobenzamido)-phenylmethyl, 4-(benzamido)phenylmethyl, 4-(benzyloxy-carbonylamino)-phenylmethyl, 4-(morpholinocarbonylamino)-phenylmethyl, 4-(Nxe2x80x2-(2-chlorophenyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(2-chlorophenyl)urea)-3-methoxyphenylmethyl, 4-(Nxe2x80x2-(2-ethylphenyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(2-isopropylphenyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(2-methoxyphenyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(2-methyl-3-pyridyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(2-nitrophenyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(2-pyridyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(2-t-butylphenyl)-urea)-phenylmethyl, 4-(Nxe2x80x2-(2-thiazolyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(3-chlorophenyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(3-methoxyphenyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(3-pyridyl)-urea)-phenylmethyl, 4-(Nxe2x80x2-(4-pyridyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(3-methylphenyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(2-methylphenyl)-urea)-phenylmethyl, 4-(Nxe2x80x2-benzylurea)-phenylmethyl, 4-(Nxe2x80x2-cyclohexylurea)-phenylmethyl, 4-(Nxe2x80x2-ethylurea)-phenylmethyl, 4-(Nxe2x80x2-isopropylurea)-phenylmethyl, 4-(Nxe2x80x2-methylurea)phenylmethyl, 4-(Nxe2x80x2-p-toluylurea)-phenyl-methyl, 4-(Nxe2x80x2-phenylurea)phenyl, 4-(Nxe2x80x2-phenylurea)phenyl-amino, 4-(Nxe2x80x2-phenylurea)phenylmethyl, 4-(Nxe2x80x2-t-butylurea)-phenylmethyl, 4-(phenylaminocarbonylamino-methyl)-phenyl, 4-(phenylsulfonamido)-phenylmethyl, 4-(t-butoxycarbonyl-amino)-phenylmethyl, 4-acetamidophenylmethyl, 4-amino-phenylamino, 4-amino-phenylmethyl, 4-benzamidophenylmethyl, 4-chlorophenylmethyl, 4-hydroxy-3-nitrophenylmethyl, 4-hydroxyphenylmethyl, 4-methoxyphenylmethyl, 4-nitrophenylamino, 4-nitrophenylmethyl, 4-phenacetamidophenylmethyl, 4-phenylphenylmethyl, 4-pyridylmethyl, 4-trifluoromethylphenylmethyl, 4-[2-(Nxe2x80x2-methylurea)-benzamido]-phenylmethyl, 4-(Nxe2x80x2-(2-methylphenyl)urea)phenylmethyl, 4-(Nxe2x80x2-phenyl-Nxe2x80x3-methylguanidino)phenylmethyl, 5-(Nxe2x80x2-phenylurea)pentyl, 5-(Nxe2x80x2-t-butylurea)-pentyl, 2,2-dimethylpropyl, 2,2-diphenylmethyl, 2,3-benzocyclobutyl, 3,4-dihydroxyphenylmethyl, 3,5-dimethoxy-4-hydroxy-phenylmethyl, 4-(1-indolecarboxylamino)-phenylmethyl, 6-methoxy-5-(Nxe2x80x2-(2-methylphenyl)urea)-2-pyridylmethyl, 4-(1,3-benzoxazol-2-ylamino)-phenylmethyl and 4-(1,3-imidazol-2-ylamino)-phenylmethyl.
Most preferably, R1 is selected from the group consisting of 4-hydroxyphenylmethyl, 3-methoxy-4-(Nxe2x80x2-phenylurea)-phenylmethyl, 4-(Nxe2x80x2-phenylurea)-phenylmethyl, 4-(Nxe2x80x2-(2-methylphenyl)urea)-phenylmethyl, 4-(Nxe2x80x2-(2-pyridyl)-urea)-phenylmethyl, 3-methoxy-4-(Nxe2x80x2-(2-methylphenyl)urea)-phenylmethyl and 6-methoxy-5-(Nxe2x80x2-(2-methylphenyl)urea)-2-pyridylmethyl.
In an alternate preferred embodiment R1 is an aryl-substituted C1-C4 alkyl group. More preferably, R1 is a (Nxe2x80x94Arxe2x80x2-urea)-para-substituted arylalkyl group. Most preferably, R1 is a (Nxe2x80x94Arxe2x80x2-urea)-para-substituted phenylmethyl group.
According to another preferred embodiment, R2 is selected from the group consisting of hydrogen, methyl and phenacyl. Most preferably, R2 is hydrogen.
According to another preferred embodiment, R3 is selected from the group consisting of 2-(methylsulfonyl)-ethyl, 3-(hyrdoxypropylthio)-methyl, 4-(methylsulfonylamino)-butyl, 4-acetylaminobutyl, aminomethyl, benzyl, butyl, hydroxymethyl, isobutyl, methyl, methylthiomethyl, phenylmethyl, propyl, 4-(benzyloxycarbonylamino)-butyl, N,N-(methylpropargyl)amino, 2-(methylthio)-ethyl, 2-(morpholino-N-carbonyl)-ethyl, 2-(N-morpholino)-ethyl, 2-(N,N-dimethylamino)-ethyl, 4-amino-butyl, 4-benzyloxyphenylmethyl, 2-benzylthiomethyl, t-butoxycarbonylaminomethyl, sec-butyl, t-butyl, N,N-dimethylaminocarbonylmethyl, 1,1-ethano*, 4-hydroxyphenylmethyl, 1-hydroxyethyl, 1-methoxyethyl, 4-methoxyphenylmethyl, benzyloxy-methyl, benzylthio-methyl, carbonylmethyl, 2-methylsulfinyl-ethyl, morpholino-N-carbonylmethyl, thiomorpholino-N-carbonylmethyl, 2-phenylethyl, asparagine side-chain, proline side-chain and 2-thiazolyl-methyl.
The amino acid side chain derived from 1-amino-cyclopropylcarboxylic acid. 
Most preferably, R3 is selected from the group consisting of isobutyl, 2-(methylthio)-ethyl, 3-(hydroxypropylthio)-methyl, 2-(methylsulfonyl)-ethyl and 4-acetylamino-butyl, 4-(methylsulfonylamino)-butyl.
According to yet another embodiment, R4 is selected from the group consisting of 4-carbomethoxyphenyl, 4-carboxyphenyl, 4-fluorophenyl, 4-methoxyphenyl, benzyl, methyl, phenyl, phenylmethyl, phenylethyl, 4-chlorophenyl, 3,4-difluorophenyl, 3,4-dimethoxyphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 4-methoxyphenyl, 2-nitrophenyl and 3-pyridyl. More preferably, R4 is selected from the group consisting 4-methoxyphenyl, 3,4-dimethoxyphenyl, 4-fluorophenyl, 4-carboxyphenyl, 4-carbomethoxyphenyl, phenylethyl and phenylmethyl.
In another preferred embodiment Y is CO, CH2 or SO2. Most preferably, Y is CO.
According to another preferred embodiment, X in formula (I) is COOH.
According to yet another preferred embodiment, n is 1.
Examples of some preferred compounds of this invention wherein X is a carboxyl group and n is 1 are provided in Table 1.
The most preferred compounds of formula (I) are: BIO-1006, BIO-1056, BIO-1089, BIO-1179, BIO-1194, BIO-1221, BIO-1224, BIO-1238, BIO-1245, BIO-1246, BIO-1248, BIO-1270, BIO-1282, BIO-1294, BIO-1321, BIO-1336, BIO-1382 and BIO-1400.
Compounds of this invention may be synthesized using any conventional technique. Preferably, these compounds are chemically synthesized from readily available starting materials, such as xcex1-amino acids. Modular and convergent methods for the synthesis of these compounds are also preferred. In a convergent approach, for example, large sections of the final product are brought together in the last stages of the synthesis, rather than by incremental addition of small pieces to a growing molecular chain.
According to one embodiment, compounds of the present invention may be synthesized in the following manner. A protected chiral amine is added to an xcex1,xcex2-unsaturated ester to produce a protected xcex2-amino acid ester. Upon suitable deprotection, the xcex2-amino acid ester is coupled to an appropriate activated ester moiety. The coupled product, if suitably functionalized, may be further reacted with yet another activated ester moiety. This material can be further manipulated to give the desired compounds of the invention. At each step of the above sequence, the ester can be hydrolyzed to the corresponding acid to give another compound of the invention.
Alternatively, the activated ester moieties mentioned above can be attached together first, then the resulting compound can be attached to the xcex2-amino acid ester portion. At this point the final manipulations and/or necessary deprotection steps can be performed.
Alternatively, under suitable conditions, the desired functionalities can be incorporated (protected or unprotected) in one of the activated ester moieties. That piece is then coupled with a xcex2-amino acid ester or a moiety consisting of a xcex2-amino ester previously coupled to an activated ester. The resulting product can then be subjected to any deprotection steps, if necessary, to give compounds of the invention.
Alternatively, the chiral xcex2-amino acid esters used in the synthesis of the compounds of this invention may be synthesized by well-known techniques, such as those described in U.S. Pat. No. 5,344,957, the disclosure of which is herein incorporated by references.
The compounds of this invention may also be modified by appending appropriate functionalities to enhance selective biological properties. Such modifications are known in the art and include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.
As used throughout this application, the term xe2x80x9cpatientxe2x80x9d refers to mammals, including humans. And the term xe2x80x9ccellxe2x80x9d refers to mammalian cells, including human cells.
Once synthesized, the activities and VLA-4 specificities of the compounds according to this invention may be determined using in vitro and in vivo assays.
For example, the cell adhesion inhibitory activity of these compounds may be measured by determining the concentration of inhibitor required to block the binding of VLA-4-expressing cells to fibronectin- or CS1-coated plates. In this assay microtiter wells are coated with either fibronectin (containing the CS-1 sequence) or CS-1. If CS-1 is used, it must be conjugated to a carrier protein, such as bovine serum albumin, in order to bind to the wells. Once the wells are coated, varying concentrations of the test compound are then added together with appropriately labelled, VLA-4-expressing cells. Alternatively, the test compound may be added first and allowed to incubate with the coated wells prior to the addition of the cells. The cells are allowed to incubate in the wells for at least 30 minutes. Following incubation, the wells are emptied and washed. Inhibition of binding is measured by quantitating the fluorescence or radioactivity bound to the plate for each of the various concentrations of test compound, as well as for controls containing no test compound.
VLA-4-expressing cells that may be utilized in this assay include Ramos cells, Jurkat cells, A375 melanoma cells, as well as human peripheral blood lymophocytes (PBLs). The cells used in this assay may be fluorescently or radioactively labelled.
A direct binding assay may also be employed to quantitate the inhibitory activity of the compounds of this invention. In this assay, a VCAM-IgG fusion protein containing the first two immunoglobulin domains of VCAM (D1D2) attached above the hinge region of an IgG1 molecule (xe2x80x9cVCAM 2D-IgGxe2x80x9d), is conjugated to a marker enzyme, such as alkaline phosphatase (xe2x80x9cAPxe2x80x9d). The synthesis of this VCAM-IgG fusion is described in PCT publication WO 90/13300, the disclosure of which is herein incorporated by reference. The conjugation of that fusion to a marker enzyme is achieved by cross-linking methods well-known in the art.
The VCAM-IgG enzyme conjugate is then placed in the wells of a multi-well filtration plate, such as that contained in the Millipore Multiscreen Assay System (Millipore Corp., Bedford, Mass.). Varying concentrations of the test inhibitory compound are then added to the wells followed by addition of VLA-4-expressing cells. The cells, compound and VCAM-IgG enzyme conjugate are mixed together and allowed to incubate at room temperature.
Following incubation, the wells are vacuum drained, leaving behind the cells and any bound VCAM. Quantitation of bound VCAM is determined by adding an appropriate colorimetric substrate for the enzyme conjugated to VCAM-IgG and determining the amount of reaction product. Decreased reaction product indicates increased binding inhibitory activity.
In order to assess the VLA-4 inhibitory specificity of the compounds of this invention, assays for other major groups of integrins, i.e., xcex22 and xcex23, as well as other xcex21 integrins, such as VLA-5, VLA-6 and xcex14xcex27 are performed. These assays may be similar to the adhesion inhibition and direct binding assays described above, substituting the appropriate integrin-expressing cell and corresponding ligand. For example, polymorphonuclear cells (PMNs) express xcex22 integrins on their surface and bind to ICAM. xcex23 integrins are involved in platelet aggregation and inhibition may be measured in a standard platelet aggregation assay. VLA-5 binds specifically to Arg-Gly-Asp sequences, while VLA-6 binds to laminin. xcex14xcex27 is a recently discovered homologue of VLA-4, which also binds fibronectin and VCAM. Specificity with respect to xcex14xcex27 is determined in a binding assay that utilizes the above-described VCAM-IgG-enzyme marker conjugate and a cell line that expresses xcex14xcex27, but not VLA-4, such as RPMI-8866 cells.
Once VLA-4-specific inhibitors are identified, they may be further characterized in in vivo assays. One such assay tests the inhibition of contact hypersensitivity in an animal, such as described by P. L. Chisholm et al., xe2x80x9cMonoclonal Antibodies to the Integrin xcex1-4 Subunit Inhibit the Murine Contact Hypersensitivity Responsexe2x80x9d, Eur. J. Immunol., 23, pp. 682-688 (1993) and in xe2x80x9cCurrent Protocols in Immunologyxe2x80x9d, J. E. Coligan, et al., Eds., John Wiley and Sons, New York, 1, pp. 4.2.1-4.2.5 (1991), the disclosures of which is herein incorporated by reference. In this assay, the skin of the animal is sensitized by exposure to an irritant, such as dinitrofluorobenzene, followed by light physical irritation, such as scratching the skin lightly with a sharp edge. Following a recovery period, the animals are re-sensitized following the same procedure. Several days after sensitization, one ear of the animal is exposed to the chemical irritant, while the other ear is treated with a non-irritant control solution. Shortly after treating the ears, the animals are given various doses of the VLA-4 inhibitor by subcutaneous injection. In vivo inhibition of cell adhesion-associated inflammation is assessed by measuring the ear swelling response of the animal in the treated versus untreated ear. Swelling is measured using calipers or other suitable instrument to measure ear thickness. In this manner, one may identify those inhibitors of this invention which are best suited for inhibiting inflammation.
Another in vivo assay that may be employed to test the inhibitors of this invention is the sheep asthma assay. This assay is performed essentially as described in W. M. Abraham et al., xe2x80x9cxcex1-Integrins Mediate Antigen-induced Late Bronchial Responses and Prolonged Airway Hyperresponsiveness in Sheepxe2x80x9d, J. Clin. Invest., 93, pp. 776-87 (1994), the disclosure of which is herein incorporated by reference. This assay measures inhibition of Ascaris antigen-induced late phase airway responses and airway hyperresponsiveness in asthmatic sheep.
The compounds of the present invention may be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained.
The compounds of the present invention may be formulated into pharmaceutical compositions that may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The term xe2x80x9cparenteralxe2x80x9d as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
The pharmaceutical compositions of this invention comprise any of the compounds of the present invention, or pharmaceutically acceptable derivatives thereof, together with any pharmaceutically acceptable carrier. The term xe2x80x9ccarrierxe2x80x9d as used herein includes acceptable adjuvants and vehicles. Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
According to this invention, the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer""s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as do natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.
The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
Alternatively, the pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols.
The pharmaceutical compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.
Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches may also be used.
For topical applications, the pharmaceutical compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with our without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic uses, the pharmaceutical compositions may be formulated in an ointment such as petrolatum.
The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation through the use of a nebulizer, a dry powder inhaler or a metered dose inhaler. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, and the particular mode of administration. It should be understood, however, that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredient may also depend upon the therapeutic or prophylactic agent, if any, with which the ingredient is co-administered.
The dosage and dose rate of the compounds of this invention effective to prevent, suppress or inhibit cell adhesion will depend on a variety of factors, such as the nature of the inhibitor, the size of the patient, the goal of the treatment, the nature of the pathology to be treated, the specific pharmaceutical composition used, and the judgment of the treating physician. Dosage levels of between about 0.001 and about 100 mg/kg body weight per day, preferably between about 0.1 and about 10 mg/kg body weight per day of the active ingredient compound are useful.
According to another embodiment compositions containing a compound of this invention may also comprise an additional agent selected from the group consisting of corticosteroids, bronchodilators, antiasthmatics (mast cell stabilizers), antiinflammatories, antirheumatics, immunosuppressants, antimetabolites, immunonodulators, antipsoriatics and antidiabetics. Specific compounds within each of these classes may be selected from any of those listed under the appropriate group headings in xe2x80x9cComprehensive Medicinal Chemistryxe2x80x9d, Pergamon Press, Oxford, England, pp. 970-986 (1990), the disclosure of which is herein incorporated by reference. Also included within this group are compounds such as theophylline, sulfasalazine and aminosalicylates (antiinflammatories); cyclosporin, FK-506, and rapamycin (immunosuppressants); cyclophosphamide and methotrexate (antimetabolites); and interferons (immunomodulators).
According to other embodiments, the invention provides methods for preventing, inhibiting or suppressing cell adhesion-associated inflammation and cell adhesion-associated immune or autoimmune responses. VLA4-associated cell adhesion plays a central role in a variety of inflammation, immune and autoimmune diseases. Thus, inhibition of cell adhesion by the compounds of this invention may be utilized in methods of treating or preventing inflammatory, immune and autoimmune diseases. Preferably the diseases to be treated with the methods of this invention are selected from asthma, arthritis, psoriasis, transplantation rejection, multiple sclerosis, diabetes and inflammatory bowel disease.
These methods may employ the compounds of this invention in a monotherapy or in combination with an anti-inflammatory or immunosuppressive agent. Such combination therapies include administration of the agents in a single dosage form or in multiple dosage forms administered at the same time or at different times.
In order that this invention may be more fully understood, the following examples are set forth. These examples are for the purpose of illustration only and are not to be construed as limiting the scope of the invention in any way.
We utilized the following procedures in the synthesis of many compounds of this invention. These procedures will be referred to by the indicated letter in the subsequent examples of the synthesis of specific compounds.
Procedure Axe2x80x94Synthesis of Cinnamate Esters
Method A: To a cinnamic acid or substituted cinnamic acid (1.0 mmol) in CH2Cl2 (10 ml) was added (COCl)2 (1.5 mmol) slowly. The reaction mixture was stirred at r.t. for 4 h and the solvent was removed in vacuo to afford the acid chloride. Methanol or t-butyl alcohol (5 ml) was added to quantitatively provide the methyl or t-butyl ester after removal of the solvents.
Method B: To an appropriate aldehyde (1.0 mmol) in THF (10 ml) was added t-butoxycarbonyl methylene triphenylphosphorane (1.0 mmol, Aldrich) and the resulting mixture was stirred at room temperature for 16 h. The reaction mixture was diluted with petroleum ether (10 ml) and was filtered through a pad of celite. The filtrate was collected and concentrated in vacuo to afford the desired product. 
Method A; Yield: 95%; (CDCl3, 300 MHz, ppm): 7.57 (d, 1H, J=16 Hz), 7.47 (m, 2H), 7.34 (m, 3H), 6.35 (d, 1H, J=16 Hz), 1.52 (s, 9H); 
Method B; Yield: 90%; (CDCl3, 300 MHz, ppm): 7.48 (d, 1H), 7.28-7.18 (m, 5H), 5.69 (d, 2H), 3.44 (d, 2H), 1.42 (s, 9H); 
Method A; Yield: 94%; (CDCl3, 300 MHz, ppm): 7.95 (d, 1H, J=16 Hz), 7.49 (d, 1H), 7.42 (t, 1H), 6.94 (dd, 2H), 6.51 (d, 2H), J=16 Hz), 3.86 (s, 3H), 3.76 (s, 3H); 
Method A; Yield: 92%; (CDCl3, 300 MHz, ppm): 7.52 (d, 1H, J=15.9 Hz), 7.28 (t, 1H), 7.09 (d, 1H), 7.02 (br, s, 1H), 6.89 (d, 1H), 6.34 (d, 1H, J=15.9 Hz) 3.82 (s, 3H), 1.54 (s, 9H); 
Method A: Yield: 98%; (CDCl3, 300 MHz, ppm): 7.64 (d, 1H, J=16 Hz), 7.29 (t, 1H), 7.10 (d, 1H), 7.06 (br, s, 1H), 6.94 (d, 1H, J=16 Hz), 3.82 (s, 3H), 3.80 (s, 3H); 
Method B; Yield: 88%; (CDCl3, 300 MHz, ppm): 8.62 (br,s, 1H), 8.51 (m, 1H), 7.72 (d, 1H), 7.48 (d, 1H, J=15.9 Hz), 7.22 (m, 1H), 6.36 (d, 1H, J=15.9 Hz), 1.49 (s, 9H); 
Method B; Yield: 90%; (CDCl3, 300 MHz, ppm): 8.60 (br, s, 1H), 7.66 (t, 1H), 7.55 (d, 1H, J=15.9 Hz), 7.36 (d, 1H), 7.21 (m, 1H), 6.78 (d, 1H, J=15.9 Hz), 1.52 (s, 9H); 
Method A; Yield: 91%; (CDCl3, 300 MHz, ppm): 7.52 (d, 1H, J=15.9 Hz), 7.44 (d, 1H, J=8.0 Hz), 6.85 (d, 1H, J=8.0 Hz), 6.21 (d, 1H, J=15.9 Hz), 3.81 (s, 3H), 1.52 (s, 9H); 
Method A; Yield: 90%; (CDCl3, 300 MHz, ppm): 7.61 (d, 1H, J=16 Hz), 7.42 (d, 2H, J=7.9 Hz), 6.86 (d, 1H, J=7.9 Hz), 6.28 (d, 1H, J=16 Hz), 3.78 (s, 3H), 3.74 (s, 3H); 
Method B; Yield: 91%; (CDCl3, 300 MHz, ppm): 7.56 (d, 1H, J=16 Hz), 7.46 (t, 2H), 7.02 (t, 2H), 6.26 (d, 2H, J=16 Hz), 1.54 (s, 9H); 
Method A; Yield: 89%; (CDCl3, 300 MHz, ppm): 7.47 (d, 1H, J=15.9 Hz), 7.01 (d, 1H, J=8.3 Hz), 6.98 (br, s, 1H), 6.78 (d, 1H, J=8.3 Hz), 3.84 (s, 6H), 1.48 (s, 9H); 
Method A; Yield: 91%; (CDCl3, 300 MHz, ppm): 7.61 (d, 1H, J=15.9 Hz, 7.07 (d, 1H, J=8.3 Hz), 7.02 (br, s, 1H), 6.83 (d, 1H, J=8.3 Hz), 6.28 (d, 1H, J=15.9 Hz), 3.88 (s, 3H), 3.76 (s, 3H); 
Method A; Yield: 92%; (CDCl3, 300 MHz, ppm):7.46 (d, 1H, J=16.1 Hz), 6.99 (s, 1H), 6.97 (d, 1H), 6.76 (d, 1H), 6.18 (d, 1H, J=16.1 Hz), 5.96 (s, 2H), 1.50 (s, 9H); 
Method A; Yield: 88%; (CDCl3, 300 MHz, ppm): 7.55 (d, 1H, J=15.9 Hz), 6.98-6.75 (m, 2H), 6.22 (d, 1H, J=15.9 Hz), 5.96 (s, 2H), 3.75 (s, 3H); 
Method B; Yield: 89%; (CDCl3, 300 MHz, ppm): 7.45 (d, 1H, J=15.8 Hz), 6.99 (s, 1H), 6.98 (d, 1H), 6.80 (d, 1H), 6.18 (d, 1H, J=15.8 Hz), 4.21 (br,s, 4H), 1.49 s, 9H); 
Method B; Yield: 88%. 
Method B; Yield: 93%; 1HNMR(CDCl3): xcex48.00(2H,d, J=5.5 Hz), 7.53 (2H, d. J=5.5 Hz), 7.58(1H,d,J=10.7 Hz), 6.42(1H,d,J=10.7 Hz), 3.90(3H, S), 1.51(9H, S).
Procedure Bxe2x80x94Synthesis of xcex2-Amino Acids
A 2 L round bottom flask, equipped with a magnetic stir bar, was charged with 1000 mL of MeOH and the flask tared with its contents. Anhydrous HCl (11 g, 0.29 mol) was bubbled in from a cylinder. To this solution was added a cinnamic acid (0.29 mol) neat in one portion. The resulting mixture was heated at reflux until the reaction was judged complete by TLC analysis. The reaction was cooled to RT, then refrigerated overnight. The crystalline product was collected by suction filtration on a medium frit and the cake washed with cold MeOH. The solid was dried on the filter to give a white or nearly white product.
Precursor to xcex2-3: Yield: 94%; TLC (3:1 hexane/EtOAc; UV): Rf=0.48; mp=134-136xc2x0 C.; 1H NMR (CDCl3, 300 MHz): 7.58 (d, 1H, J=15.9 Hz), 7.00-6.97 (m, 3H), 6.79 (d, 1H, J=7.9 Hz), 6.24 (d, 1H, J=15.9 Hz), 5.98 (s, 2H), 3.77 (s, 3H); MS (FAB): 206.
Precursor to xcex2-5: Yield: 84%; TLC (3:1 hexane/EtOAc; UV): Rf=0.48; mp=89-91xc2x0 C.; 1H NMR (CDCl3, 300 MHz): 7.63 (d, 1H, J=15.9 Hz), 7.46 (d, 2H, J=8.7 Hz), 6.89 (d, 2H, J=8.7 Hz), 6.29 (d, 1H, J=15.9 Hz), 3.82 (s, 3H), 3.77 (s, 3H); MS (FAB): 192.
Michael Addition of (R)-(+)-N-benzyl-1-phenylethylamine to Methyl 4-methoxy-cinnamate
A 1 L 3-neck round bottom flask, equipped with a stopper, thermometer, and 250 mL addition funnel with an Ar inlet was charged with (R)-(+)-N-benzyl-1-phenylethylamine hydrochloride (0.132 mol, 32.6 g, 1.1 eq based on cinnamate) and the apparatus flushed with Ar 30 min. The salt was suspended in dry THF (200 mL) and the mixture cooled to an internal temperature of xe2x88x9270xc2x0 C. with a dry ice/acetone bath. To the suspension was added n-BuLi (2.5 M in hexanes, 0.257 mol, 103 mL, 1.95 eq based on amine hydrochloride) from the addition funnel at such a rate that the internal temperature did not exceed xe2x88x9265xc2x0 C. The addition required 90 min. After completing the addition, the reaction was stirred at xe2x88x9270xc2x0 C. for 1 hr. A solution of methyl 4-methoxycinnamate (0.120 mol, 23 g, 1 eq) in THF (125 mL) was added from the addition funnel over 90 min at such a rate that the internal temperature did not exceed xe2x88x9265xc2x0 C. After completing the addition, the reaction was stirred at xe2x88x9270xc2x0 C. 2 hrs. TLC analysis indicated complete reaction. The reaction was quenched cold by the addition of 5% citric acid (250 mL) and stirred overnight at RT. In a 2 L separatory funnel, the layers were separated and the organic washed with 5% citric acid (1xc3x97125 mL). The combined aqueous were extracted with EtOAc (1xc3x97200 mL). The combined organics were then washed with 5% NaHCO3 (1xc3x97150 mL) and brine (1xc3x97150 mL) and dried (MgSO4). Filtration and evaporation to constant weight provided crude product (50.04 g, 103% of theory) as a viscous oil which solidified on standing. Pure material was obtained by triturating and stirring crude product with heptane (1.5-2 mL/g, 75-100 mL total volume) at RT overnight. The solids were collected by suction filtration on a medium frit and the cake washed by flooding with cold heptane (2xc3x9750 mL). The solids were dried on the filter to give pure product (28.93 g, 60% yield) as a white powder. TLC (4:1 hexane/EtOAc): Rf=0.50 (I2, UV); mp=87-88xc2x0 C.; 1H NMR (CDCl3, 300 MHz): 1.20 (d, 3H, J=6.9 Hz), 2.51 (dd, 1H, J=9.4, 14.8 Hz), 2.66 (dd, 1H, J=5.7, 14.8 Hz), 3.45 (s, 3H), 3.67 (ABq, 2H, J=14.7 Hz), 3.79 (s, 3H), 3.98 (q, 1H, J=6.8 Hz), 4.37 (dd, 1H, J=5.7, 9.3 Hz), 6.86 (d, 2H, J=8.6 Hz), 7.16-7.33 (m, 10H), 7.40 (d, 2H, J=7.3 Hz); MS (FAB): 404
Hydrogenolysis of Benzyl Groups
The above adduct (0.071 mol, 28 g) was suspended in MeOH (300 mL) and treated with formic acid (96%, 0.179 mol, 8.25 g, 6.8 mL, 2.5 eq) neat in one portion with stirring. To this suspension was added Degussa type E101 NE/W 10% Pd/C (50% wet, 0.00179 mol, 3.81 g, 0.025 eq) in one portion. The resulting mixture was heated at reflux for 1-2 hr until judged complete by TLC analysis. The mixture was cooled to RT, then filtered on a pad of Celite, washing the flask and pad with MeOH (150 mL). The combined filtrates were evaporated to give crude product (15.42 g, 102% of theory) as an oil. The crude product was dissolved in i-PrOH (250 mL) and heated to a gentle reflux. D-tartaric acid (0.071 mol, 10.76 g, 1 eq) was added as a solid in one portion. Heating was continued for 15 min, during which time the salt precipitated as a fine white solid. The mixture was cooled to RT, then refrigerated overnight. The crystalline salt was collected by suction filtration on a medium frit, washing with cold i-PrOH (50-75 mL), and dried on the filter to give product (23 g, 79%). The above salt was converted to the free base by dissolving in a minimum volume of H2O (125 mL) and treating the solution with solid NaHCO3 until the aqueous was saturated. This was extracted with EtOAc (3xc3x97100 mL). The combined organics were washed with brine (1xc3x97100 mL) and dried (MgSO4). Filtration and evaporation provided pure product (11.75 g, 78%) as a nearly colorless oil which solidified on cooling. TLC (9:1 CHCl3/MeOH): Rf=0.30 (I2, UV); HPLC (reverse phase; MeCN/H2O/TFA gradient): 96% pure, Rt=17.9 min; 1H NMR (CDCl3, 300 MHz): 1.87 (br s, 2H), 2.62 (d, 2H, J=6.9 Hz), 3.64 (s, 3H), 3.76 (s, 3H), 4.35 (t, 1H, J=6.9 Hz), 6.84 (d, 2H, J=8.6 Hz), 7.25 (d, 2H, J=8.6 Hz); MS (FAB): 210. 
1H NMR (CDCl3, 300 MHz, ppm) 6.81 (d, 1H, J=1.6 Hz), 6.72 (d, 1H, J=7.9 Hz), 6.66 (d, 1H, J=7.9 Hz), 5.85 (s, 2H), 4.22 (1H, dd, J=7.5 Hz and 7.3 Hz), 2.47 (2H, dd, J=7.5 Hz and 5.6 Hz), 2.21(s, 2H), 1.35 (9H, s). 
1H NMR (CDCl3, 300 MHz, ppm) 6.82 (d, 1H, J=1.6 Hz), 6.76 (d, 1H, J=7.9 Hz), 6.73 (d, 1H, J=7.9 Hz), 5.89 (s, 2H), 4.29 (1H, dd, J=6.9 Hz and 6.8 Hz), 3.63 (3H, s), 2.57 (d, 2H, J=6.9 Hz), 1.75 (s, 2H); 
1H NMR (CDCl3, 300 MHz, ppm) 6.79-6.78 (m, 3H), 4.32 (t, 1H, J=6.7 Hz), 3.75 (s, 3H), 3.72 (s, 3H), 2.52 (d, 2H, J=6.8 Hz), 1.82 (br, 2H), 1.42 (s, 9H).; 
1H NMR (CDCl3, 300 MHz, ppm) 7.20 (d, J=8.6 Hz), 6.80 (d, 2H, J=8.6 Hz), 4.30 (t, 1H, 6.8 Hz), 3.71 (s, 3H), 3.60 (s, 3H), 2.57 (d, 2H, J=6.8 Hz), 1.91 (s, 2H); 
1H NMR (CDCl3, 300 MHz, ppm) 7.24 (d, J=8.4 Hz), 6.82 (d, 2H, J=8.4 Hz), 4.26 (t, 1H, 6.8 Hz), 3.66 (s, 3H), 2.47 (d, 2H, J=6.6 Hz), 1.41 (s, 9H); 
1H NMR (CDCl3, 300 MHz, ppm) 7.21(dd, 1H, J=8.2 Hz and 8.1 Hz), 6.95-6.93 (m, 2H), 6.78 (d, 1H, 6.8 Hz), 4.34 (t, 1H, J=6.7 Hz), 3.79 (s, 3H), 2.54 (d, 2H, J=6.9 Hz), 1.74 (s, 2H), 1.40 (s, 9H); 
1H NMR (CDCl3, 300 MHz, ppm): 7.34-7.08 (m, 2H), 6.82-6.68 (m, 2H), 4.45 (m, 1H), 3.65 (s, 3H), 3.49 (s, 3H), 2.58 (d, 2H), 1.68 (br s, 2H). 
1H NMR (CDCl3, 300 MHz, ppm) 7.28-7.25 (m, 2H), 7.01 (d, 1H), 4.31(t, 1H), 2.50 (d, 2H), 2.01 (br, 2H), 1.41 (s, 9H); 
1H NMR (CDCl3, 300 MHz, ppm) 6.84 (s,1H), 6.79-6.76 (m, 1H), 4.24-4.19 (m, 1H), 4.19 (s, 4H), 2.50 (d, 2H), 1.63 (br, 2H), 1.41 (s, 9H); 
1H NMR (CDCl3, 300 MHz, ppm) 3.34-3.05 (m, 1H), 2.65-2.58 (m, 2H), 1.65 (d, 2H) 1.45 (fs, 9H); 
1H NMR (CDCl3, 300 MHz, ppm) 7.34-7.28 (m, 3H), 7.26-7.15 (m, 3H), 3.42-3.15 (m, 1H), 2.71 (dd, 1H, J=5.5 Hz and 13.3 Hz), 2.54 (dd, 1H, J=8.1 Hz and 13.3 Hz), 2.36 (dd, 1H, J=4.2 Hz and 15.7 Hz), 2.20 (dd, J=8.6 and 15.7 Hz), 1.42 (s, 9H).
To prepare xcex2-13 amino acid 
1M TMSCl in CH2Cl2 (33 ml, 33 mmol) was added to a mixture of (R)-xcex1-methylbenzylamine (3.4 g, 28 mmol) and Et3N (4 g, 40 mmol) in THF (10 ml) was added and the mixture was allowed to stir for 1 h at room temperature. After the solid was removed by filtration, the solution was concentrated to afford a liquid. This silylamine (2.4 g, 12.5 mmol) was dissolved in THF (35 ml) and was cooled to xe2x88x9278xc2x0 C. To this cooled solution was added n-BuLi (7.8 ml of 1.6 M solution in hexanes, 12.5 mmol) slowly. After stirring for 0.5 h at the temperature, to the reaction mixture was added a solution of t-butyl trans-3-(3-pyridyl)acrylate (2.56 g, 12.4 mmol) in THF (10 ml). The stirring was continued for another xc2xd h and the mixture was quenched with sat. NH4Cl (20 ml) and was allowed to warm up to room temperature and extracted with ether. The combined ether layers were dried (K2CO3) and concentrated to afford an oil. This oil (500 mg) was dissolved in ethanol (1.5 ml), t-butanol (15 ml), ammonium formate (1.5 g) and 10% Pd/C (1.2 g) were added. The resulting mixture was heated to reflux for 3 h followed by acid and base workup to afford the desired amine xcex2-13 (300 mg). FAB-MS=223.
1HNMR(CDCl3): xcex47.97(2H, d, J=5.4 Hz), 7.41(2H, d, J=5.4 Hz), 4.40(1H, t, J=4.5 Hz), 3.88(3H,S), 2.55(2H, d, J=4.5 Hz), 1.71(2H, br), 1.39(9H, S).
General Procedure for Synthesis of M-1, M-2 and M-3
To a solution of the commercially available amino acid (1.5 mmoles) in CH2Cl2 (4 ml) and MeOH (1 ml) cooled to 0xc2x0 C., was added thionyl chloride (0.125 ml, 1.65 mmol). The reaction was warmed to 40xc2x0 C. for 2 h, and concentrated to dryness in vacuo to afford the desired amino ester HCl salt. 
89% yield; 1HNMR (DMSO-d6, 300 MHz, ppm): 9.00-8.75 (3H, bm), 7.71 (2H, d, J=7.3 Hz), 7.58 (2H, d, J=7.3 Hz) 4.71 (1H, bs), 3.64 (3H,s), 3.40-3.06 (2H, m); 
85% yield as a tan solid. 1HNMR (CDCl3, 300 MHz, ppm): 7.55-7.05 (6H, bm), 3.66 (3H, s), 3.65-3.45 (2H, bm), 3.10-2.77 (5H, bm), 2.17-1.95 (2H, bm); 
84% yield as a pale tan solid; 1HNMR (CDCl3, 300 MHz, ppm): 8.1-7.8 (4H, bm), 7.65-7.45 (3H, bm), 5.45 (1H, br), 3.80-3.30 (2H, bm), 3.55 (3H, s).
Proceudre Cxe2x80x94Synthesis of Coupled Amino Acids
To a solution of ethyl 3-amino-3-phenyl-1-propanoate (or other xcex2-amino acid ester prepared by Procedure B) (0.50 g, 5.25 mmol) in CH2Cl2 (5 ml) was added BocLeuOSu (1.5 g, 4.67 mmol) (CbzLeuOSu is used for the Cbz protected analog) with cooling and Et3N (5 drops). The mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with CH2Cl2 (10 ml) and washed with 5% citric acid (5 mlxc3x972), 5% NaHCO3 (5 ml) and sat. NaCl (5 ml). The organic layer was dried (Na2SO4) and concentrated to afford 1.26 g (66%) as a white solid.
Procedure Dxe2x80x94Synthesis of Deprotected Amino Acids
To a stirred solution of the product of Procedure C (a Boc-Leu-xcex2-amino acid ester) (41.5 mg, 0.102 mmol) at 0-5xc2x0 C. in 2 mL of CH2Cl2 was added 4 mL of TFA. The mixture was allowed to come to room temperature with continued stirring for 1 hour. The reaction was concentrated in vacuo, redissolved in CH2Cl2, concentrated two more times and placed under high vacuum to remove final traces of TFA. HPLC showed complete conversion to two new peaks of shorter retention time. The residue can taken up in DMF and TEA added with stirring until basic to litmus in preparation for further reaction. A Cbz group is removed using the following method:
The product from Procedure C (where t-butyl 3-amino-3-phenyl-1-propanoate and CbzLeuOSu were used) (110 mg, 0.23 mmol) in MeOH with a catalytic amount of 10% palladium on charcoal was stirred overnight under hydrogen at 40 psi. The reaction was filtered through Celite(copyright) and concentrated in vacuo yielding the free base Leu-xcex2-amino acid ester (87 mg, quantitative) as a clear oil. 1H NMR: (CDCl3, 300 MHz, ppm), 7.30 (m, 5H), 5.33 (dd, 1H, J=6, 8.82 Hz), 4.00 (m, 1H) 2.77 (dd 1H J=9, 15 Hz), 2.90 (dd, 1H, J=6, 15 Hz), 1.69 (m, 2H), 1.45 (m, 1H), 1.29 (s, 9H), 0.90 (d, 6H, J=6 Hz).